The objective of this project is to use a cell-free DNA synthesis system prepared from bacteriophage T7 infected E. coli to investigate the molecular mechanism of DNA replication. In particular I wish to investigate in detail the mechanism of initiation of DNA chains during replication. My approach will emphasize studies with purified proteins known from genetic analysis to participate in replication in vivo. The phage gene 4 protein will be purified to homogeneity using an in vitro complementation assay. The molecular weight, subunit composition, and other physical properties of this protein will be determined. The protein will be assayed for a variety of catalytic activities related to DNA synthesis. Preliminary studies using a partially purified preparation of gene 4 protein suggest that the protein greatly stimulates the synthesis of DNA off a duplex T7 DNA template by the T7 DNA polymerase. The requirements for this reaction will be studied in detail and the DNA product will be analyzed by a variety of techniques. I will determine whether an RNA "primer" is covalently attached to the 5' terminus of the DNA product. The specificity of initiation will be investigated using a type II restriction endonuclease. Finally I will search for factors present in T7 infected cells which will further stimulate DNA synthesis in this purified system. If such factors are identified they will be purified and studied.